1. Field of the Invention
This invention relates to regulation of cell growth, and more particularly to regulation of cancer cell growth. In particular, peptides and polypeptides derived from particular regions of the syndecan 4 molecule has been shown to inhibit engagement of α6β4 integrin by EGFR, thereby limiting tissue invasion and tumor cell growth and/or survival.
2. Related Art
It is well established that the EGFR family of receptor tyrosine kinases, in particular EGFR and HER2, have major roles in human cancer (Hynes and Lane, 2005; Hynes and MacDonald, 2009; Scaltriti and Baselga, 2006), notably breast cancer, head and neck squamous cell carcinoma (HNSCC), and lung cancer for EGFR. EGFR is a causal agent in triple negative (ER-, PR-, HER2-) breast cancer (also classified as “basaloid”), which comprises 15-25% of breast cancers (Herschkowitz et al., 2007; Livasy et al., 2006; Perou et al., 2000; Teng et al., 2011). The triple negative (TN) tumors are especially malignant, often strike younger women (especially African-Americans) and are resistant to tamoxifen therapy (Livasy et al., 2006; Diaz et al., 2005; Haupt et al., 2010; Lu et al., 2008; Carvalho et al., 2010; Friedrichs et al., 1995), the common mode of treatment for most women. These women usually present as node-positive at first diagnosis and often die of metastatic disease (Dent et al., 2007; Haffty et al., 2006). EGFR has also long been recognized to play a central role in progression of squamous cell carcinoma of the head and neck (H&N), in which it is overexpressed in over 80% of the tumors, and the negative response of H&N patients to therapy (Chang and Califano, 2008; Perez-Ordonez et al., 2006). It is commonly hyperactivated by overexpression of its ligands (e.g., EGF, TGFα), by activating EGFR mutations, and by its overexpression (Chung et al., 2004; Grandis et al., 1997, Cassell and Grandis, 2010. Furthermore, γ-irradiation, the treatment of choice for many tumors, leads to EGFR activation by a variety of mechanisms (Zimmerman et al., 2006)—including increased EGFR expression (Schmidt-Ullrich et al., 1994), inhibition of phosphatases that would otherwise maintain receptor quiescence, and promoting the shedding (e.g., activation) of the membrane-anchored pro-forms of EGFR ligands (Dent et al., 1999)—leading to significantly poorer 5-yr survival rates in patients with EGFR-positive tumors.
The α6β4 integrin and its ligand LN332 are upregulated in breast and squamous cell carcinomas, and are linked to invasion, metastasis and recurrent disease (Choi and Chen, 2005; Ginos et al., 2004; Patarroyo et al., 2002; Wilhelmsen et al., 2006). Identified as the TSP-180 antigen in mouse tumors (Falcioni et al., 1986), or the A9 antigen in humans (Van Waes et al., 1991; Kimmel and Carey, 1986), high expression of this antigen predicts a higher rate of early relapse in H&N cancer patients (Wolf et al., 1990; Carey et al., 1987). In more recent studies exploring its tumor-promoting role using animal models, keratinocytes that lack expression of the β4 integrin subunit fail to form invasive squamous cell carcinomas when transformed with ras and IκB, unlike their normal counterparts that express the integrin (Dajee et al., 2003; Tran et al., 2008). Despite this seeming importance of the integrin in squamous cell carcinoma, there currently are no therapeutics available to target its tumor-promoting activities.
A number of labs have investigated the potential role of syndecans in α6β4-mediated cell migration and tumorigenesis. But these studies have focused largely on syndecans acting as co-receptors with the α6β4 integrin in laminin binding rather than a role in directly regulating α6β4 activation. The phosphorylated and “activated” α6β4 integrin redistributes to the leading edges of invading keratinocytes or tumors; these leading edges overexpress the “unprocessed” form of LN332 that retains the LG4,5 heparin-binding region that engages syndecans (Amano et al., 2000; Marinkovich et al., 1992; Matsui et al., 1995; Goldfinger et al., 1999; Goldfinger et al., 1998). Interestingly, recent work from Rouselle's group shows that Sdc1 and Sdc4 bind differently to the LG4,5 domain and speculates that this may account for somewhat different cell behaviors mediated by these two syndecans (Carulli et al., 2012). Other work shows that expression of LG4,5 supports tumorigenesis in an animal model of squamous cell carcinoma, again suggesting a potential role for syndecans in tumorigenesis (Tran et al., 2008), although it is admittedly indirect.
Although not widely appreciated, the α6β4 integrin is also expressed on vascular endothelial cells in vivo, where its function in hemidesmosomes allows the endothelium to resist frictional forces as it does on stratified epithelia. Giancotti has shown a clear role for α6β4 integrin in tumor angiogenesis and that α6β4 is expressed in the vasculature of several tumor types (prostate, breast, glioma, papillary thyroid, melanoma) (Nikolopoulos et al., 2004). Although not studied extensively, it clear that endothelial cells express EGFR family members (Amin et al., 2006) and that tumor endothelial cells upregulate the expression of EGFR in particular (Amin et al., 2006; Bohling et al., 1996; Bruns et al., 2000; Kedar et al., 2002; Huang et al., 2002). Despite this seeming importance of the integrin in squamous cell carcinoma and tumor-induced angiogenesis, there currently are no therapeutics available to target its tumor-promoting activities.
Work from a variety of laboratories has shown a linkage between the α6β4 integrin and EGFR in breast and other cancers (Lu et al., 2008; Folgiero et al., 2008). This integrin in normal cells assembles with laminin in the basement membrane underlying basal epithelial cells as well as endothelial cells lining blood vessels, forming stable hemidesmosomes in which the long (ca. 1000 amino acid) cytoplasmic domain of the β4 subunit anchors to the keratin filament network in the cytoplasm of the cell (Wilhelmsen et al., 2006; Hopkinson and Jones, 2000; Nievers et al., 1999). In contrast to this “stabilizing” role, however, the integrin takes part in the invasion, proliferation and survival of tumors that overexpress the receptor tyrosine kinases HER2, EGFR, or c-Met—leading to the assembly of these kinases with the integrin (Wilhelmsen et al., 2006; Agazie and Hayman, 2003; Mainiero et al., 1996; Mariotti et al., 2001; Bertotti et al., 2005; Bertotti et al., 2006; Bon et al., 2007; Falcioni et al., 1997; Gambaletta et al., 2000; Santoro et al., 2003; Trusolino et al., 2001; Tsuruta et al., 2008; Giancotti, 2007). There is evidence for this in TN breast tumors, especially, as α6β4 integrin and EGFR overexpression are causally linked in this disease and lead to poor prognosis (Lu et al., 2008). When coupled with the integrin, signaling from these kinases disrupts the hemidesmosome (Rabinovitz et al., 2004; Wilhelmsen et al., 2007) and leads to tyrosine phosphorylation of the β4 cytoplasmic domain, providing docking sites for signaling effectors that drive tumor cell proliferation, invasion and survival (Wilhelmsen et al., 2006; Mariotti et al., 2001; Bertotti et al., 2006; Wilhelmsen et al., 2007; Mainiero et al., 1997; Shaw et al., 1997; Guo et al., 2006; Merdek et al., 2007; Dutta and Shaw, 2008; Datta et al., 1999; Dans et al., 2001; Shaw, 2001; Yang et al., 2010). The distal third of the β4 tail containing these phosphorylation sites has thus been termed the β4 “signaling domain” (Guo et al., 2006) (FIG. 1). In studies using the MMTV-Neu mouse model of HER2+ breast cancer, replacement of native β4 with a β4 mutant (β1355T) lacking this signaling domain acts as a suppressor of breast cancer (Guo et al., 2006), suggesting that the wild type β4 receptor normally couples with HER2 to drive tumorigenesis of HER2+ breast cancer as well. Work utilizing a number of mammary carcinoma cell lines, focusing mostly on HER2+ cells, also shows that HER2/α6β4 signaling is critical for invasion and survival of these tumors (Falcioni et al., 1997; Gambaletta et al., 2000; Guo et al., 2006). Complementing their expression in the tumors, HER2 and EGFR are also expressed in endothelial cells, especially those induced by tumors (Amin et al., 2006; Bruns et al., 2000; Kedar et al., 2002), and couple with the α6β4 integrin during tumor-induced angiogenesis (Nikolopoulos et al., 2004).